Indicators for detecting the presence of metabolic byproducts from microorganisms

ABSTRACT

Polymeric indicator films and pH indicating wraps are provided for visually monitoring, detecting, and/or determining the presence of metabolic byproducts from harmful or potentially harmful microorganisms. Also provided are methods of use and preparation of the polymeric indicator films.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/693,375, filed Jan. 25, 2010; which claims the benefit under 35U.S.C. §119(e) to U.S. provisional application Ser. Nos. 61/147,450,filed Jan. 26, 2009; 61/258,515, filed Nov. 5, 2009; 61/269,961, fieldJun. 30, 2009; and 61/297,234, filed on Jan. 21, 2010. All of which areincorporated hereby by reference in their entirety.

TECHNICAL FIELD

This invention relates generally to polymeric indicator films useful,for example, in pH indicating food wraps and wound dressings, Thisinvention is also directed to uses of these films for detecting thepresence of bacterial growth as measured by bacterial growthby-products. Such by-products alter the pH of an aqueous composition incontact with the film.

BACKGROUND

The presence of undesirable bacterial contamination in food productsintended for consumption is of significant concern to manufacturers,farmers, packagers, food distributors, wholesalers, retailers,consumers, and to worldwide public health. A particularly worrisomeconcern is bacterial contamination in packages containing food productsfor human consumption. The United States boasts of the safest food inthe world; however, each year approximately one in four individualssuffer from a food borne illness and some 5,000 die from something theyhave eaten. According to the Center for Disease Control and Prevention,each year in the United States, 76 million people contract some kind offood borne illness, 325,000 are hospitalized and 5,000 fatalities occurdue to contamination of consumed food. In Third World countries it hasbeen estimated that bacterial contaminated food and water kills over twomillion children each year. Despite those numbers, most food borneinfections are undiagnosed and unreported.

Packaging of perishable and edible food products may be susceptible toundesired and undetectable bacterial growth during each stage in thefood chain from harvest to consumption. Minimal levels of bacterialcontamination (bacterial load) of food is deemed acceptable in food forconsumer use. Indeed, regulatory agencies such as the FDA haveestablished limits on bacterial load permitted in the food.Nevertheless, it is very hard to determine if bacterial growth in foodalters the bacterial level of the food to unacceptable levels. Foodinitially safe for consumption may be altered by undetected bacterialgrowth due to poor handling, improper storage and other factors. At allpoints in the food chain, it would be of great benefit if there was anunmistakable means to determine that there has been unacceptablebacterial growth occurring on the food.

Still further, bacterial contamination of wounds can lead to seriousinfection, illness, and even death if the contamination is unnoticed anduntreated for even a relatively short period of time. Often times,bacterial infection is first detected by the presence of inflamed redskin around a wound site. Visualization of the wound by skin redness isoften at a point where the infection has significantly progressed withinthe diseased patient.

Examples of such wounds are those generated by use of central venouscatheters, cannulae, and related medical devices (hereafter “catheters”)which are inserted and maintained through the skin. As is apparent,catheters are used on a variety of patients, usually in a hospitalsetting. These catheters provide secure access e.g., into a patient'sblood vessel and allow for the safe administration of fluids and drugsinto the patient or the removal of fluids from the body.

Wounds of all nature carry an inherent risk of bacterial infections. Inaddition to intentionally created wounds such as those described above,other wounds susceptible to infection include abrasions, burns, surgicalincisions, injection sites, and the like.

For example, catheter insertion into the body can cause seriouscomplications. Specifically, catheter related bloodstream infection(CR-BSI) is a serious and potentially life-threatening complication whencatheter insertion sites into blood vessel lumen become infected withbacterial microorganisms. Conventional state of the art care nowrequires that these insertion sites be covered with a wound dressing asa preventive measure against such infections.

A number of factors render such insertion sites especially susceptibleto bacterial contamination. Specifically, the catheter essentiallycompromises the skin's natural protective barrier, providing a directroute to bypass the body's first line of immunity. In addition, uponinsertion into the host, the outer surface of the catheter is quicklycovered with host proteins that facilitate bacterial attachment andgrowth. There is also evidence that implanted abiotic material itselfcauses local attenuation of antimicrobial immune responses, therebyinhibiting a normal immune response against bacterial biofilm formation.Finally, patients who possess the greatest need for catheterization areoften immunologically compromised and are therefore more susceptible tobacterial infection.

Catheters themselves are generally infected via one of two generalroutes, typically by microorganisms that compromise the natural florasurrounding the site of catheter insertion. For example, bacteria maycontaminate the catheter along its outer surface, and it is believedthat this type of infection often occurs during the initial insertion ofthe catheter through the skin. Catheters can also be contaminated intheir lumenal compartments where fluids flow from contaminated infusatesolutions. The most prevalent bacteria found to be the cause ofbacterial sepsis are from the exterior flora surrounding the insertionsite.

Catheter-related bloodstream infections are notoriously difficult totreat via conventional antibiotic therapy, with associated mortalityrates ranging from 12% to 25%. Catheter related bloodstream infection isthe most frequent serious complication seen with catheters withinfections occurring in as many as 3% to 7% of all catheter placements,which is estimated to affect more than 250,000 patients in U.S.hospitals each year. In addition, these infection complications extendhospital stays, necessitate active intervention on the part ofhealthcare personnel, and result in driving the estimated annualdomestic healthcare cost associated with complications arising fromthese catheter-related infections to more than nine billion dollars.

The use of a wound covering (sometimes referred to as a “dressing” or“wound dressing”) in conjunction with a catheter is conventional butdoes not entirely obviate the underlying infection risk as evidenced bythe statistics above. Such wound dressings are typically placedproximate the catheter injection site and contact fluids exuding fromthat site.

Still further, other wounds such as burns, abrasions, surgicalincisions, and the like are particularly susceptible to infection. Inhospital settings, infections caused by antibiotic resistant bacteriasuch as Staphylococcus is a major concern and a cause of morbidity.

Therefore, a need exists for methods and medical devices and woundcoverings for the detection of bacterial growth contamination in orabout a wound that can readily detect and indicate the presence ofmicroorganisms well before the infection has progressed to the pointthat it manifests itself by skin redness.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to a polymeric indicator filmcomprising a transparent and uncolored polymer layer or layers and aplurality of pH indicating moieties, wherein the pH indicating moietiesare entrapped within the polymer layer or between two or more of thepolymer layers and further wherein the pH indicating moieties retain thetransparency and lack of color of the polymer film at neutral pH butwhich impart color to at least a portion of the film when exposed to anacidic pH.

In another aspect, this invention provides a “sandwich” type ofpolymeric indicator film comprising:

a) a first layer comprising one or more hydrophilic, hydronium ionpenetrating layers;

b) a second layer comprising one or more hydrophobic, water impermeablelayers; and

c) a pH indicator layer placed between the first layer and second layer.

In some embodiments, the polymeric indicator film is for detecting thepresence of bacterial growth in food. In some embodiments, the polymericindicator film is for detecting the presence of bacterial growth in oraround a wound.

In some embodiments, the pH indicating moieties are selected fromheptamethoxy red and hexamethoxy red or a combination thereof. Theseindicators are particularly useful as when used in an amount to detectpH change in either food or bodily fluids, they retain the transparentand uncolored nature of the polymeric indicator film at a neutral pH.However, when the pH becomes acidic (due to by-products of bacterialgrowth), the color of the indicator film becomes red. This permits readydetermination that bacterial growth has occurred.

In another aspect, there is provided a process for preparing a polymericindicator film comprising a first hydrophilic, hydronium ion penetratingtransparent layer and a second hydrophobic, water impermeabletransparent layer which process comprises:

-   -   a) selecting one or more hydrophilic, hydronium ion penetrating        layers as the first layer of the polymeric film wherein the        first layer has a front and back surface;    -   b) selecting one or more hydrophobic, water impermeable layers        as the second layer of the polymeric film wherein the second        layer has a front and back surface;    -   c) applying a pH indicator layer to at least a portion of one        surface of the second layer; and    -   d) bonding the first and second layers together such that the pH        indicator layer is placed between the first and second layers,        wherein the pH indicator layer comprises hexamethoxy red and/or        heptamethoxy red or derivatives thereof.

Other embodiments of the invention are further described in the DetailedDescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one example of a polymeric indicator film of thisinvention.

FIG. 2 illustrates an exploded side view of a wound dressing inaccordance with an embodiment of the invention.

FIG. 3 illustrates a top view of the sheet liners 102 and 110 inaccordance with an embodiment of the present invention.

FIG. 4 illustrates a first example of a process for preparing thepolymeric indicator film of FIG. 1.

FIG. 5 illustrates a second example of a process for preparing thepolymeric indicator film of FIG. 1.

It should be appreciated that like reference numerals are used toidentify like elements illustrated in one or more of the figures. Itshould also be appreciated that the figures may not be necessarily drawnto scale.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the text refers to various embodimentsrelating to compounds, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather, it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

DEFINITIONS

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise.

The term “comprising” is intended to mean that the compounds and methodsinclude the recited elements, but not excluding others. “Consistingessentially of” when used to define compositions and methods, shall meanexcluding other elements of any essential significance to the compoundsor methods. “Consisting of” shall mean excluding more than traceelements of other ingredients for claimed compounds and substantialmethod steps. Embodiments defined by each of these transitional termsare within the scope of this invention. Accordingly, it is intended thatthe processes and compositions can include additional steps andcomponents (comprising) or alternatively include additional steps andcompounds of no significance (consisting essentially of) oralternatively, intending only the stated methods steps or compounds(consisting of).

The term “polymeric indicator film” refers to a polymeric filmcomprising one or more pH indicator moieties incorporated therein whichis able to indicate the presence of a threshold level of bacterialby-products by a visible color change. In some embodiments, thepolymeric indicator film comprises a hydrophilic, hydronium ionpenetrating layer and a hydrophobic, water impermeable layer. In someembodiments, the hydrophilic, hydronium ion penetrating layer contains aplurality of pH indicator moieties embedded therein. In someembodiments, the pH indicator moieties are positioned between thehydrophobic, water impermeable layer and the hydrophilic, hydronium ionpenetrating layer optionally by use of an adhesive.

Neutral pH has a value of 7.0. As used herein, the term “neutral pH”also includes low acid pH of from about 5 to below 7 and low basic pH offrom above 7 to up to about 8.

The term “acidic” as used herein refers to an acidic pH range generallyproduced from by-products of bacterial growth. Such acidic pHs generallyrange from above 1 to about 5 and, preferably, a pH range of from 2 toabout 5. A strong acid has a pH of below 2.0.

The term “transparent layer” refers to a polymer layer which issufficiently transparent to visible light that a viewer can readily seethrough the layer. Preferably, the transparent layer is uncolored (asthat term is defined below).

The term “hydrophobic, water impermeable layer” refers to a transparentpolymer layer wherein the polymer is hydrophobic and does not permitwater, water vapor, bacteria or protons (hydronium ions) to readilypenetrate into or through the layer. Especially preferred hydrophobic,water impermeable layers are those which qualify as food grade and/ormedical grade polymers. In some embodiments, the hydrophobic, waterimpermeable polymer is polyethylene (PE). In some embodiments, thatpolymer includes, but is not limited to, polyvinylalcohol, polyethyleneterephthalate, poly(vinylidene fluoride), poly(vinyl chloride),poly(vinylidene chloride), polypropylene, phenoxy resins,butadiene/styrene copolymers, butadiene/methylstyrene copolymers,poly(meth)acrylates, butadiene/acrylonitrile copolymers,ethylene/propylene copolymers, polybutadiene, polyisoprene,poly(oxy-2,6-dimethyl-1,4-phenylene),poly(oxycarbonyloxy-1,4-phenyleneisopropylidene-1,4-phenylene),acrylonitrile styrene copolymers, acrylonitrile/methylacrylate/butadiene copolymers, acrylonitrile/styrene/butadienecopolymers, poly-1-vinylnaphthalene, polyvinylphenyl ketone,poly-p-xylenedodecanedioate, poly-tetramethylene octenediamide,poly-tetramethylene terephthalene, poly-trimethylene-3,3′-dibenzoate,poly-terephthallic anhydride, poly-4-methyl-diamine, polyvinylenecarbonate, polyvinylene laurate, polyisopropenyl acetate,polyallylbenzene, polyvinylbutyl ether, polyvinyl formate, polyvinylphenyl ether, polynorbornadine, polycarbonate, hydrophobic polyestersand polyurethanes, or a mixture thereof.

The term “barrier membrane” is synonymous with “hydrophobic, waterimpermeable layer” and is sometimes used herein relative to the polymerlayer over a wound dressing to prevent water or water vapor or bacteriafrom penetrating through that layer from the environment and contactingthe wound site.

The term “hydrophilic, hydronium ion penetrating layer,” which issometimes referred to herein as a “hydrophilic, hydronium ion permeablelayer,” refers to a transparent polymer layer wherein the polymer ishydrophilic and permits water, water vapor, gases and protons (hydroniumions) to diffuse into or through this polymeric layer which causesincrease of the hydronium ion concentration and decrease of the pH.Proton penetration can be determined by a number of measures but is mosteasily measured by the use of a pH indicator which detects the presenceof a sufficient number of protons by a color change. Examples ofhydrophilic, hydronium ion penetrating transparent layers includeseveral which are commercially available materials having high moisturevapor transmission rate, such as polyether block amide copolymers (e.g.,Pebax®). Preferred hydrophilic, hydronium ion penetrating polymer layersare those which qualify as food grade and/or medical grade polymers. Insome embodiments, the hydrophilic, hydronium ion penetrating uncoloredtransparent layer comprises a polymer selected from the group consistingof polyether block amide (e.g., Pebax®), (poly)hydroxyethylmethacrylate, (poly)hydroxypropyl methacrylate, (poly)glycerolmethacrylate, copolymers of hydroxyethyl methacrylate, hydroxypropylmethacrylate or glycerol methacrylate and methacrylate acid,aminoacrylate and aminomethacrylate, (poly)vinyl pyrrolidone,(poly)vinylpyridine, polar polyamides, methyl cellulose, hydroxypropylcellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, ethylhydroxyethylcellulose, carboxymethyl cellulose, cellulose acetate,cellulose acetate butyrate, cellulose acetate propionate, cellulosenitrate, polyvinyl acetate, polyvinyl alcohol, copolymers of polyvinylacetate and polyvinyl alcohol, hydroxyl modified copolymers of vinylacetate and vinyl chloride, polyesters, polyurethanes containing atleast about 10% by weight of polyethylene oxide, styrene/methacrylicacid/hydroxyethyl methacrylate copolymers, styrene/methacrylicacid/hydroxypropyl methacrylate copolymers,methylmethacrylate/methacrylic acid copolymers, ethylmethacrylate/styrene/methacrylic acid copolymers, ethylmethacrylate/methyl methacrylate/styrene/methacrylic acid copolymers,polytetrafluoroethylene and hydrophilic cellulose copolymers.

The term “permeable membrane” is synonymous with “hydrophilic, hydroniumion penetrating transparent layer” as defined above and is sometimesused to describe the polymer layer of, for example, the wound dressingor the sheath overlaying a catheter at the catheter insertion site thatis in contact with body fluids at that site. Permeable gases include butnot limited to oxygen, carbon dioxide, carbon monoxide, hydrogensulfide, hydrogen, sulfur dioxide, and ammonia among others. Thepermeability of the permeable membrane is such that it permits asufficient concentration of pH altering gases to diffuse into or throughthis polymeric layer so as to sufficiently alter the pH and produce avisual colorimetric reaction with the indicator. In some embodiments,the permeable membrane comprises one or more hydrophilic, hydronium ionpenetrating transparent layers described above.

The term “bonding” refers the formation of a single polymeric film byadhering two or more separate layers into a single film by conventionaltechniques.

The term “colorless” refers to the lack of sufficient color so as to bedeemed transparent and clear either visually or by instrumentation. Whenvisually evaluated, the term “colorless” does not mean that there is nocolor but, rather, the color is either not visually detectable orminimally detectable such that the viewer sees a clear film.

The term “colorful” refers to sufficient color such that the color canbe detected by visual observation.

The term “threshold level of bacterial by-products” refers to the amountof by-products produced by bacteria such that the pH changessufficiently to effect a change in the color of the indicator fromcolorless to colorful. Preferably, the threshold level of bacterialby-products is a level at or below the level produced by a minimumamount of bacteria growth that would cause concern when present on foodor at a wound or catheter insertion site.

The term “indicator” refers to a substance capable of changing colorwith a change in pH caused when a threshold amount of bacterialby-products are produced. In one embodiment, the indicator is a pHindicator. Such pH indicators are sometimes referred to herein as “pHindicating moieties”. Bacterial by-products include, but are not limitedto, gaseous carbon dioxide, hydrogen sulfide, sulfur dioxide, hydrogen,ammonium, lactate, and mixtures thereof. Mixtures of these by-productswith moisture result in the formation of acids such as carbonic acid,sulfuric acid, ammonium hydroxide, lactic acid, or mixtures thereof.When a sufficient amount of acid is generated, the indicator produces achange from uncolored to colored that is readily discernable by even anuntrained observer.

Examples of pH indicators include xylenol blue(p-xylenolsulfonephthalein), bromocresol purple(5′,5″-dibromo-o-cresolsulfonephthalein), bromocresol green(tetrabromo-m-cresolsulfonephthalein), cresol red(o-cresolsulfonephthalein), phenolphthalein, bromothymol blue(3′,3″-dibromothymolsulfonephthalein), p-naphtholbenzein(4-[alpha-(4-hydroxy-1-naphthyl)benzylidene]-1(4H)-naphthalenone),neutral red (3-amino-7-dimethylamino-2-methylphenazine chloride),hexamethoxy red and heptamethoxy red, and combinations thereof.

In a preferred embodiment, the pH indicators are hexamethoxy red and/orheptamethoxy red or derivatives thereof.

The term “bacteria” as used herein refers to any bacteria that may bepresent in the either food or a wound site regardless of origin and thatmay further be a potential health hazard. Bacteria detectable by thepolymeric indicator films provided herein include Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus mitis, Streptococcus sanguis,Enterococcus faecium, Escherichia coli, Enterobacter cloacae,Enterobacter aerogenes, Enterococcus faecalis, Pseudomonas aeruginosa,Klebsiella pneumonia, Salmonella, Candida albicans, gram negativebacilli, or a combination thereof.

The term “catheter” includes any and all known catheters which puncturethe skin and are used for delivering fluids, medicaments, etc. into thebody, assisting in the elimination of fluids from the body, and/or fordiagnostic purposes. Such catheters include central venous catheters,diagnostic catheter, drainage catheters, and the like. Also includedwithin the term “catheter” are cannulae which are conventional, wellknown, tubes inserted into the body by puncture through the skin, forthe delivery or removal of fluids. Cannulae normally come with a trocarwhich permits puncturing of the body.

The term “food spoilage” refers to the growth of microorganisms, such asbacteria, on food. As used herein, rancidity, which is a breakdown ofthe cellular matrix of the tissue or meat via protein denaturizationprocess and release of proteins (enzymes) to the extracellular spaces ofthe tissue, is not detected by the invention.

Polymeric Indicator Films

This invention provides a polymeric indicator film comprising atransparent and an uncolored polymer layer or layers and a plurality ofpH indicating moieties, wherein the pH indicating moieties are entrappedwithin the polymer layer or between one or more of the polymer layersand further wherein the pH indicating moieties retain the transparencyof the uncolored polymer film at a neutral pH but which impart color toat least a portion of the film when exposed to an acidic pH.

In some embodiments, the polymeric indicator film is for detecting thepresence of bacterial growth in food. In some embodiments, the polymericindicator film is for detecting the bacterial growth in or around awound or a catheter insertion site. In particular, one or moreembodiments of the invention provide for polymeric indicator films whichare colorless in the absence of a threshold level of bacterial growthand colorful when bacterial growth exceeds such a threshold both ofwhich are determined by the generation of by-products of bacterialgrowth which alter the color of the indicator film.

In another aspect, this invention provides a polymeric indicator filmfor visually detecting the growth of bacteria, said polymeric indicatorfilm comprising a hydrophilic, hydronium ion penetrating polymer layerand a plurality of pH indicating moieties selected from heptamethoxy redand hexamethoxy red or a combination thereof, wherein the pH indicatingmoieties are entrapped within the polymer layer. These polymericindicator films can be employed in situations where the monitoring ofbacterial growth is desirable, such as in food or medical settings.

Heptamethoxy red refers to the chemical2,4,6,2′,4′,2″,4″-heptamethoxytri-phenylcarbinol having Formula (I)below and derivatives thereof which do not alter the ability of thecompound to detect changes in pH. Hexamethoxy Red refers to the chemical2,4,2′,4′,2″,4″-hexamethoxytriphenylcarbinol having Formula (II) belowand derivatives thereof which do not alter the ability of the compoundto detect changes in pH.

Heptamethoxy red exhibits a dynamic range between approximately pH 7 andpH 5, and hexamethoxy red exhibits a dynamic range between approximatelypH 4.5 and pH 2.6. Both exhibit visually perceptible color change fromcolorless to colorful (reddish/violet red) when exposed to a sufficientamount of acid so as to lower the pH to a level necessary to effectcolor change. Derivatives of either hexamethoxy red or heptamethoxy redare contemplated to include substitution of one or more of the methoxygroups with a C₂-C₃ alkoxy group provided that such substitution doesnot alter the ability of the compound to detect changes in pH.

In some embodiments, the pH indicating moieties are in both gaseous andfluid communication with the bacterial by-products and acids producedtherefrom.

In some embodiments, the pH indicating moieties are entrapped within thepolymeric film and typically within the hydrophilic, hydronium ionpenetrating layer of the polymeric film. General methods for entrapmentinclude those described in U.S. Pat. No. 5,629,360. The pH indicatingmoieties may also be entrapped within pores in the hydrophilic,hydronium ion penetrating layer. Suitable additives such as diatomaceousearth (DE), TiO₂, and SiO₂ or combinations thereof may be added to thelayer to generate such pores. In some embodiments, the diatomaceousearth is in an amount of up to about 10% w/w. In some embodiments, it isabout 4% w/w. In some embodiments, it is about 3% w/w. Pores may also begenerated by laser drilling. Weight percents are based on the weight ofthe additive relative to the weight of the hydrophilic, hydronium ionpenetrating layer.

The pH indicating moieties in the polymeric indicator film are employedin an amount effective for detecting a color change thereby evidencing achange in pH. As used herein, the term “detection” denotes acolor-change either visible by human eye having ordinary vision.Instrumentation may also be used. In some embodiments where the pHindicating moiety is embedded in the hydrophilic, hydronium ionpenetrating layer, the pH indicating moiety is employed in an amount ofabout 0.01% w/w to about 10% w/w relative to the weight of that layer.In some embodiments the pH indicating moiety is employed in an amount ofabout 1% w/w to about 3% w/w.

In other embodiments, provided is a “sandwich” type of polymericindicator film comprising:

-   -   a) a first layer comprising one or more hydrophilic, hydronium        ion penetrating layers;    -   b) a second layer comprising one or more hydrophobic, water        impermeable layers; and    -   c) a pH indicator layer placed between the first layer and the        second layer.

In some embodiments, the pH indicator layer comprises a sufficientamount of pH indicator moieties to provide visible color change in atleast a portion of the polymeric pH indicator film upon contact withbacterial growth by-products.

In some embodiments, the pH indicator layer has a thickness of from 200Angstroms to 5 microns.

In some embodiments, the polymeric indicator film further comprises anadhesive layer placed between the first hydrophobic, water impermeablelayer and the pH indicator layer.

The adhesive layer may be any adhesive suitable for adhering the pHindicator(s) to the surface thereof, so that the pH indicator(s) form aseparate layer over the adhesive. Preferably the adhesive is non-toxic(more preferably food grade safe) when dried and has a neutral orslightly basic pH, for example a pH not significantly above the pKa ofthe pH indicator applied, so that it does not interfere with the abilityof the pH indicator to change color in the presence of bacterial growth.In some embodiments, the adhesive is a medical grade adhesive when thepolymeric indicator is for medical application. In some embodiments, theadhesive is an adhesive that meet all standards as set forth by the FDAfor food contact and/or food additives. In some embodiments, theadhesive is used in an amount such that the resulting polymeric filmretains an uncolored, transparent nature at neutral pH.

FIG. 1 illustrates an example of the “sandwich” type of polymericindicator film of this invention. In particular, polymeric indicatorfilm 1 comprises an outer barrier layer polymer 3 which is ahydrophobic, water impermeable polymer as described herein. The outerlayer 3 comprises a first and second surface, 3 a and 3 b respectively.In one embodiment, an adhesive layer 5 is applied to the second surface3 b of outer barrier layer 3. Application of the adhesive layer 5 can beconducted in any manner known in the art and the specific means forapplying such a layer is not part of the invention. In one embodiment,the adhesive layer 5 can be applied by spraying an adhesive solventsystem onto the second surface 3 b. In another embodiment, the adhesivelayer 5 can be applied by solvent casting wherein the solution of theadhesive and the solvent are applied to surface 3 b (facing upward) anda conventional spreader is used to spread a uniform thickness of thesolution to that surface followed by partial or complete drying of thesolvent. Indicator layer 7 is applied in a manner likewise to that ofadhesive layer 5 albeit each layer may be applied in the same ordifferent manner. For example, indicator layer 7 can be applied bysolvent casting and adhesive layer 5 can be applied by spraying. Theinner or inside barrier layer 9 is then attached to the indicator layer7 in such a manner that a cohesive laminated polymer indicator film 1 isformed. “Cohesive” as used herein means that visually, the laminatedpolymeric layered film will not readily separate and is viewed as asingle film. Such polymeric indicator films can be prepared by theprocesses described in details below. It is understood that the terms“inner” and “outer” are used solely to differentiate the two layers. Inone embodiment, the “inner” layer can be placed adjacent a food productsuch as raw meat such that the fluids from the meat contact thehydrophilic, hydronium ion penetrating layer of the film whereas the“outer” layer interfaces the environment where the meat is stored and asthe outer layer is water impermeable, it retards water penetrationthrough the film. In another embodiment such as when used as a cathetersheath, the “outer” layer may interface with the catheter surface whilethe “inner layer” will interface with the bodily fluids.

In one embodiment, the pH indicator is selected from the groupconsisting of hexamethoxy red and heptamethoxy red, and combinationsthereof.

The pH indicating moieties detect pH change associated with by-productsof bacterial growth. These by-products include, among others, gaseouscarbon dioxide, hydrogen sulfide, sulfur dioxide, hydrogen, ammonium,lactate, and mixtures thereof. Mixtures of the by-product with moistureresult in the formation of acids such as carbonic acid, sulfuric acid,ammonium hydroxide, lactic acid, or mixtures thereof that react with theindicator to produce a color change. The term “by-products” withreference to bacteria, refer to the gases that are expelled from thebacteria due to their natural growth of populations in numbers. Suchgases can be in the vapor state or can combine with water or behydrolyzed to form an acid such as sulfuric acid, carbonic acid,hydrogen sulfide or other gaseous or water vapor state which lowers thepH of the immediate environment with increasing concentrations of thegas vapor or water vapor combination.

In some embodiments, the acid is generated from a bacteria or is formedby reaction of a bacterial by-product with water, said bacterialby-product is selected from the group consisting of carbon dioxide andsulfur dioxide.

It is contemplated that in addition to bacteria, microbes detectable bythe packaging materials include, among others, viral and fungalmicrobes. Among bacteria whose growth in food can be detected by themethods described herein include but are not limited to Bacillus,Brucella, Campylobacter, Clostridium, Escherichia coli, Listeriamonocytogenes, Salmonella, Streptococcus, Pseudomonas, Staphylococcus,Shigella spp., Vibrio spp., Yersinia spp., coliform or spore formingbacteria and other food borne pathogens known to be involved in foodcontamination or a mixture of such microbes. Particular strains havebeen identified as associated with fresh vegetables. For example,Escheria coli O157:H7 was associated with prepackaged spinach:“Investigation of an Escheria coli O157:H7 Outbreak Associated with DolePre-Packaged Spinach,” California Food Emergency Response Team FinalReport, Mar. 21, 2007 (available from the California Department ofHealth Services, Food and Drug Branch, P.O. Box 997435, MS 7602,Sacramento, Calif. 95899-7435 and also available from U.S. Food and DrugAdministration San Francisco District, 1431 Harbor Bay Parkway, Alameda,Calif. 94502.)

Food Spoilage Adaptation

The polymeric indicator films of this invention can be used on food orin bags or containers into which foods are placed to detect the presenceof metabolic byproducts from bacteria.

Accordingly, in another aspect of this invention, provided is a foodstorage container containing a polymeric indicator film of thisinvention. In some embodiments the food storage container is a sealablebag. In other embodiments the food storage container is a jug or bottlefor storing liquids.

In some embodiments, the polymeric indicator film is laminated. In thisaspect of the present invention, the general approach to producing alaminate film is easily recognized and very well-known by those ofordinary skill in the art. One of ordinary skill may readily modify theteachings of the present specification to produce laminates comprised ofnumerous layers which will fall within the scope of the presentinvention.

In some embodiments, provided is a flexible wrap comprising an outerhydrophobic, water impermeable layer, an indicator layer wherein theindicator layer is as described herein and an inner hydrophilic,hydronium ion permeable layer. In some embodiments, the outer layercomprises one or more hydrophobic, water impermeable layers as describedherein. In some embodiments, the hydrophobic, water impermeable outerlayer of the polymeric indicator film is resistant to the passage ofenvironmental and ambient gaseous compounds such as oxygen, hydrogen,nitrogen, moisture or other elements.

In some embodiments, provided is a pH indicating film or flexible wrapcomprising an outer hydrophobic, water impermeable layer and an innerhydrophilic layer permeable to microbial byproducts and acids thereof,wherein sandwiched between the outer and inner layers is a plurality ofpH indicating moieties selected from heptamethoxy red and hexamethoxyred or a combination thereof, and wherein the pH indicating moieties areentrapped within a polymer or are adherent to an adhesive, said pHindicating moieties and optionally the adhesive being situated betweenthe outer and inner layers.

In some embodiments, the inner layer is permeable to microbialbyproducts but impermeable to molecules having a molecular weight ofabout 200 daltons or more. Suitable inner barrier layers includepolymeric films such as those found in Tegaderm™ (3M, St. Paul Minn.).In some embodiments, the inner barrier layer comprises one or morehydrophilic, hydronium ion penetrating layers described herein.

In some embodiments, outer layer comprises a hydrophobic, waterimpermeable layer described herein. In some embodiments, an adhesivelayer is present on one surface of the outer barrier layer and thepolymeric indicators are immobilized on the surface of the adhesive. Inother aspects, the adhesive is a transparent, colorless (in the amountused) food safe adhesive. Suitable adhesive include Elmer's sprayadhesive.

Suitable outer layers include polyethylene films, e.g., Glad® wrap orSaran™ wrap.

Suitable outer layers include the polyurethane films and films such asthose used in Tegaderm™.

In some embodiments, the polymeric indicator film or flexible wrap hasincorporated into, attached thereto or printed thereon a machinerecognizable code such as a barcode or a RFID (radio frequencyidentification) tag.

Food or food stuff refers to any edible substance including solids andliquids such as meats, fish, vegetables, milk, milk products such asyogurt, cottage cheese, ice cream, etc., fruit and the like. Preferably,the food used in combination with the polymeric indicators of thisinvention are those which, when contaminated by microbes, provide for adetectable byproduct either from the food or the microbe that alters thepH of the food in a detectable manner.

As an alternative to an adhesive layer, the pH indicator can be castonto the hydrophobic, water impermeable layer and/or the hydrophilic,hydronium ion penetrating layer using any number of different coatingtechnologies, including: air knife coating, curtain coating, gap coating(knife over roll, knife over blanket, floating knife, etc.), gravurecoating (engraved roll, offset engraved roll), immersion (dip) coating,mayer bar (meyer bar, metering rod, wire wound rod), reverse rollcoating (L-head, nip-fed, pan-fed), rotary screen, or slot die (slot,extrusion). The choice of which coating technology might be selected tocreate the film is determined, in part, by the desired characteristicsof the resulting film (i.e., film thickness). Other factors such asviscosity, surface tension, dry speed, production costs, etc., can alsohave a bearing on selection of which coating technology might beselected to create the film.

In some embodiments, the pH indicating moieties are placed as a separatelayer between the hydrophilic, hydronium ion penetrating layer and thehydrophobic, water impermeable layer. In some aspects of theseembodiments, the layer of pH indicating moieties is placed only over aportion of the film (for example) in the form of a warning (DO NOT EAT)which would be generated by the change in pH.

For the commercial practicability, a meyer bar coating technology may beemployed where a 2 to 5 mil wet film thickness, and preferably, a 4.0mil wet film thickness of the adhesive solution is applied to apolyethylene film.

A variety of configurations of the polymeric indicator film and pHindicating flexible wraps are provided. In one embodiment, the packagingmaterial may be in the form of a roll of film that can be dispensed froma cardboard box having a cutting edge top and allowing for sheets ofvarying lengths to be dispensed and used for covering of certainproducts. In other embodiments the packaging material is dispensed froma large roll with heat sealability. In still other embodiments, thepackaging material is in the form of a container or “baggie” with aresealable side or top and is used for storage of certain food products.Another configuration for the packaging material, a strip or polymersheet or card comprising the polymeric indicator is inserted into thecontainer.

For food spoilage, use of the indicators provided is based on theconcept of pH change caused by the presence of bacterial metabolicby-products. The pH change can be caused by numerous sources, including:gases, liquids containing electrolytes, ions and molecules thatinfluence pH like lactic acid, citric acid and ammonia. As thedefinition of pH is the negative log of the hydrogen ion concentration,used to express the acidity or alkalinity of a solution, moieties whicheffect this ionic concentration change may be detectable.

Some embodiments of the invention relate to the detection of by-productsof contaminating bacterial growth in a packaged food product to providean early warning of possible microbial growth occurring during storagein that package. These food products may be within the group commonlyknown as the low acid foods comprising meats, poultry, dairy, seafoodand the like. These low acid foods have an inherent pH of near neutralor pH 7 or between pH 7.4 and 6.2. Foods known to be within the classreferred to as medium acid foods are soups and pasta and have aninherent pH of 4.5 to 5.0. Foods that are known to be within the classreferred to as acid foods are fruits and vegetables with an inherent pHbetween 3.7 and 4.5. Food known to be within the class referred to ashigh acid foods include lemons and pickled products with an inherent pHof between 2.3 and 3.7. In certain embodiments, food products other thanthose within the low acid range that have a more acidic characteristicmay not be included in the applicable food product packaging for usewith certain embodiments of this invention when the inherent lower pHvalues of the foods cause a reaction with the pH indicator of thepackaging material and signal a false-positive result.

As hexamethoxy red and heptamethoxy red have significantly differentpKa's (they change colors at different pHs), it is within the skill ofthe art to select the appropriate indicator relative to the acidity ofthe food stored within the polymeric indicator wrap of this invention.

Accordingly, in some embodiments provided is a method for detectingwhether food is spoiled or contaminated by bacterial growth such thatsaid food is not edible, said method comprising:

-   -   a) placing a portion of said food proximal to the polymeric        indicator film or pH indicating wrap;    -   b) detecting the presence or absence of a colorimetric change in        the polymeric indicator film or pH indicating wrap; and    -   c) correlating the presence or absence of a colorimetric change        in the polymeric indicator or pH indicating wrap to whether the        food is non-edible or edible.

Medical Device Adaptation

In another aspect, provided are polymeric indicator films and pHindicating wraps for visually detecting bacterial growth in a medicalsetting, for example, related to wound dressings or catheter insertionsites. The presence of bacterial metabolic by-products is detected by achange in the color of the polymeric indicator film or pH indicatingwraps positioned at or proximal to the site of wounds or catheterinsertion sites.

In one aspect, the invention relates to wound dressings and methods fortheir use in detecting the by-products produced by bacterial growth. Insome embodiments, the wound dressing comprises a polymeric indicatordescribed herein.

In one embodiment, the wound dressing comprises indicators which areassociated with the dressing in a manner to prevent leaching of theindicators from the dressing.

In one embodiment, the indicators are embedded in the wound dressing ina manner such that the indicator does not leach from the wound dressing.For example, the indicators can be placed as a layer sandwiched betweenthe hydrophilic, hydronium ion penetrating polymer layer and thehydrophobic, water impermeable polymer layer.

The skin is the largest organ of the human body. One of the keyfunctions that the skin performs is to protect the body's “insides” fromthe external environment by acting as a barrier and/or a filter betweenthe “outside” and the “inside” of the body. The skin has other functionssuch as regulating the body's temperature and allowing for the excretionof some selected body wastes and toxins.

The acid/base balance is very important to metabolic health and plays avery important role in human physiology. The measure of acids and basesis conducted by determining the pH level, which is the inverse log ofthe hydrogen ion concentration. The pH scale or range is between 0 and14 with 7 being neutral. Acids range between pH 0 to less than pH 7 andbases from above pH 7 to pH 14. pH 7 is defined as neutral—neitheracidic nor basic. Weak acids are between pH 5.5 and less than pH 7 andweak bases between above pH 7 and pH 8.5.

The human skin pH is affected by numerous endogenous factors such asmoisture, sweat concentrations, sebum, genetic predisposition and age.The skin contains pores, which are a combination of oil and sweat(sebaceous and sudoriferous) glands that assist in keeping the skinhealthy. Skin secretions are a result of sweat or sebum secretion. Inadolescents, there are often increased levels of sebum oil secretionstimulated by sex hormones. In general the normal excretion of oil andsweat from the skin's pores maintains a slightly acidic condition ofapproximately pH 5.5. The pH of normal, healthy surface skin is betweenpH 4.5 and pH 6. Some researchers have reported normal, healthy skin pHto be as low as pH 4 and as high as pH 6. Thus, the literature hosts arange for normal healthy skin pH values.

The skin acts as a protective mantel for the body and is sometimesreferred to as the “acid mantel”. The skin is the first defensemechanism against bacteria as the acidic skin condition provides anunfavorable environment for bacterial growth. This acidic defense varieswith age. Typically newborns have a skin pH closer to neutral (pH 7)that quickly turns slightly acidic in order to protect the skin frombacterial growth. In the late teens and early 20's the acid mantlebecomes well developed and provides an even better and more acidicdefense against potentially harmful, external environmental factors.With the onset of old age the human skin can become more neutral in itspH and more susceptible to bacterial growth.

Differences in skin pH values have been evaluated in different races andgender. No significant differences have been reported between males andfemales although females were found to have a slightly lower skin pHthan men. African-Americans were found to have a slightly more acidicstratum corneum (surface layer) than their Caucasian counterparts withrespect to age. There is little variation in surface skin pH from onesite on the body to another.

Provided are wound dressings comprising a polymeric indicator filmdescribed herein that are operational at skin pH and provide a colorchange, for example, colorless to colorful transition, which allows forfacile visual detection of microbial contamination.

In one embodiment, provided is a wound dressing which comprises:

-   -   a) a polymeric film having a top and bottom surface, said bottom        surface for facing a wound site susceptible to bacterial        contamination, which film comprises a hydrophilic, hydronium ion        penetrating layer, a plurality of pH indicating moieties for        indicating the presence of the bacterial growth by-products,        said moieties being associated with or embedded within the        hydrophilic, hydronium ion penetrating layer and said moieties        change from colorless in the absence of a threshold level of        bacterial by-products to colorful in the presence of a threshold        level of bacterial by-products; and    -   b) a hydrophobic, water impermeable layer adjacent to the top        surface of said polymeric film.

In another embodiment, the indicating moieties are pH indicators and,preferably, are either hexamethoxy red or heptamethoxy red, morepreferably, hexamethoxy red.

In another embodiment, the indicating moieties are associated with saidpolymeric film by forming a separate layer over the hydrophilic,hydronium ion permeable layer.

In another embodiment, the indicating moieties are associated with saidhydrophilic, hydronium ion permeable layer by embedding said moietieswithin said layer such that said moieties are not capable of leachingtherefrom.

In one embodiment, the pH indicating moieties are covalently bound tothe hydrophilic, hydronium ion permeable layer.

The pH indicating moieties can be covalently linked, for example, to thehydrophilic, hydronium ion permeable film by coupling the functionalgroups on the pH indicators or introduced onto the indicators tocomplementary functional groups present on the film under reactionconditions suitable for forming the desired bonds. The pH indicators canalso be synthetically modified to display the desired functional groupfor coupling to the polymer such as an aldehyde or an acyl group.Likewise, a polymeric surface can also be modified to present thedesired functional group for coupling to the pH indicator. Anotherapproach may be to sequester the pH indicator moieties within the matrixof the polymeric film, yet allowing the functionality of the pHindicator to remain intact.

-   -   In some embodiments, provided is a wound dressing which        comprises:    -   a) an inner layer comprising one or more hydrophilic, hydronium        ion penetrating layers;    -   b) an outer layer comprising one or more hydrophobic, water        impermeable layers; and    -   c) a pH indicator layer placed between the inner layer and outer        layer.

Uses of the wound dressings provided herein include placement proximateto wound sites susceptible to bacterial contamination in a mannerwherein the hydrophilic, hydronium ion permeable layer is capable ofcontacting bodily fluids.

Wound sites susceptible to contamination by microorganisms includingskin wounds, abrasions, burns, openings, surgical incision sites,puncture sites, and catheter insertion sites containing, for example,central venous catheter or other catheter used for insertion into thelumen of an artery or vein.

In one embodiment, provided is a method for detecting the presence ofcontaminating bacterial at a site susceptible to bacterialcontamination, comprising covering the site with the wound dressingsdisclosed herein and monitoring the dressing for a color change.

By empirical data, growth of certain bacteria under controlledconditions may be correlated to pH such that the wound dressingsdescribed herein may be calibrated for different sensitivities (e.g., toshow a colorimetric change at an earlier time) or for different types ofbacteria.

An example of a wound dressing is shown in FIGS. 2 and 3 havingindicator layer 120 between a barrier membrane 114 and optionally apermeable membrane 118. An adhesive 116 is provided around thecircumference of barrier membrane 114. Optionally, the wound dressingcomponents can also be packaged between sheet liners 102 and 110 tofacilitate their application to the skin.

In one example, barrier membrane 114 is a transparent, hydrophobic, andpolymeric barrier membrane that acts as a barrier to the outsideenvironment. Water, water vapor, and/or bacterial growth are preventedfrom penetrating to the wound or catheter insertion site from theenvironment by barrier membrane 114. Barrier membrane 114 does permitthe passive diffusion of water vapor and oxygen from under the wounddressing or catheter insertion site to the environment. Barrier membrane114 further permits the passive diffusion of oxygen from the environmentthrough the barrier membrane to the skin and creates a moist environmentat the surface of the skin and wound or catheter insertion site whilelimiting water vapor loss from the underlying tissue.

Barrier membrane 114 includes an adhesive 116 that is used to contactthe skin and make a secure bond that is a perimeter of adhesionsubstantially along the border of barrier membrane 114. Adhesive 116 isa medical grade adhesive along the circumference/border of barriermembrane 114 and prevents the wound dressing from being dislodged orinadvertently removed from the skin.

Permeable membrane 118 is beneath (i.e., closer to the wound or catheterinsertion site or skin) barrier membrane 114 and, in one example, is atransparent, hydrophilic polymeric membrane. Permeable membrane 118 doesnot extend fully to the margins of barrier membrane 114 but is held inplace by adhesive 116 used to secure the wound dressing and indicator tothe skin. Permeable membrane 118 is permeable to gases, water vapor, andgases dissolved in water vapor in one example. In yet another example,permeable membrane 118 is permeable to gases that include, oxygen,carbon dioxide, carbon monoxide, hydrogen sulfide, hydrogen, sulfurdioxide, lactic acid and ammonia among others, such that theconcentration of gas which may ultimately diffuse through the polymericcomposition is sufficient to produce a visual colorimetric reaction withindicator layer 120 that is easily visualized through barrier membrane114, thereby alerting a user, such as an attending healthcareprofessional, of potentially harmful bacterial growth beneath the wounddressing at the catheter insertion site.

The polymers in the wound dressings disclosed herein can comprisesubstantial quantities of monomers having polar groups associated withthem, such that overall polymeric composition is rendered hydrophilic.Preferably, the polymeric compositions are comprised of monomers whichcontain for example, hydroxyl groups, ester groups, amide groups,urethane groups, or carboxylate groups. While not being limited by wayof theory, it is believed that the inclusion of polar groups allowswater to more readily permeate the polymer and consequently, bringdissolved gases into proximity of the indicator contained within the twomembranes and evoke a visible color change reaction.

Membranes 114 and 118 may be chemically/physically functionalized (e.g.,to include different functional exchange groups with different backbone)to allow for selective control over passage through the membrane (e.g.,to allow specific molecules to pass and/or for molecules to pass in aspecific direction (e.g., away from or toward the wound site)).

The wound dressings can be packaged in various ways and in oneembodiment is packaged similarly to a dressing apparatus trademarked asTegaderm™, available from 3M Health Care Ltd., of St. Paul, Minn.Optionally, barrier membrane 114, indicator layer 120, and permeablemembrane 118 may be packaged between sheet liners 102 and 110 tofacilitate application of the wound dressing and indicator to thesurface of the skin over the site of the indwelling central venous orother catheter.

In one example, sheet liner 102 is a thin and rigid sheet of thin cardon the barrier side or top side of barrier membrane 114, with “wings”104 at each end. Sheet liner 102 may include a window 106 pre-cut suchthat window 106 may be peeled away to reveal barrier membrane 114underneath, thus leaving barrier membrane 114, indicator layer 120, andpermeable membrane 118 suspended on a frame of sheet liner 102, whichfacilitates precise placement of the film and reduces wrinkling.Accordingly, window 106 may be peeled away and removed just prior toapplication of the film to the skin and allows for visualization of thecatheter insertion site through the two transparent membranes 114 and118 and indicator layer 120. Sheet liner 102 may also include a slit 108along one side of the border of sheet liner 102 to allow for the peelingaway of the border after the wound dressing is firmly applied to theskin at the site of the catheter insertion.

In a further example, sheet liner 110 is printed sheet of release papercoupled to the adhesive 116 adjacent to permeable membrane 118. Sheetliner 110 may also have wings 112 extending beyond the wings 104 ofsheet liner 102 to aid in application to the skin. Sheet liner 110 isremoved just after window 106 of sheet liner 102 is removed, and thewound dressing applied to the skin over the catheter site.

It will be apparent that the elements of the dressing that contact theskin, including but not limited to the barrier membrane, the adhesive,and the permeable membrane, are composed of medical grade materials andin one example meet the requirements for long-term skin contact asestablished by the United States Food and Drug Administration.

Indicator Preparation

A synthesis of heptamethoxy red or hexamethoxy red and a propheticpreparation of the polymeric indicator is shown in the Examples.

Preparation of the Polymeric Indicator Films

In another aspect provided herein is an improved method for preparingthe pH indicating moieties according to the procedures disclosed herein.

In some embodiments, there is provided a process for preparing apolymeric indicator film comprising an inner hydrophilic, hydronium ionpenetrating transparent layer and an outer hydrophobic, waterimpermeable transparent layer which process comprises:

-   -   selecting one or more hydrophilic, hydronium ion penetrating        layers as the inner layer of the polymeric film wherein the        inner layer has a first and second surface;    -   selecting one or more hydrophobic, water impermeable layers as        the outer layer of the polymeric film wherein the outer layer        has a first and second surface;    -   applying a pH indicator layer to at least a portion of one        surface of the outer layer; and bonding the inner and outer        surfaces together such that the pH indicator layer is placed        between the inner and outer layers.

In some embodiments, the process further comprises applying an adhesivelayer to the one surface of the outer layer on which the pH indicatorlayer will be applied, which step is performed prior to application ofthe pH indicator layer.

In some embodiments, there is provided a process for preparing apolymeric indicator film comprising an inner hydrophilic, hydronium ionpenetrating transparent layer and an outer hydrophobic, waterimpermeable transparent layer which process comprises:

-   -   a) selecting one or more hydrophilic, hydronium ion penetrating        layers as the inner layer of the polymeric film wherein the        inner layer has a first and second surface;    -   b) selecting one or more hydrophobic, water impermeable layers        as the outer layer of the polymeric film wherein the outer layer        has a first and second surface;    -   c) applying an adhesive layer to one surface of the outer layer;    -   d) applying a coating of pH indicator to at least a portion of        the adhesive layer; and    -   e) bonding the inner and outer layers together such that the        adhesive layer coated with the pH indicator is placed between        the inner and outer layers, thereby forming the polymeric        indicator film.

In some embodiments, the adhesive is partially dried before the coatingof pH indicator is applied. In some embodiments, the outer layer withthe indicator applied thereto is dried before binding of the outer layerwith the inner layer.

In some embodiments, the pH indicator layer comprises a sufficientamount of pH indicator moieties to provide visible color change in atleast a portion of the polymeric pH indicator film upon contact withmicroorganism growth.

In some embodiments, the pH indicator layer has a thickness of fromabout 200 Angstroms (Å) to about 5 microns.

In the manufacture process, the outer or inner film layers, preferablythe outer layer, would serve as the base on a rolling stock productionassemble and would feed through a line in which the adhesive spray isapplied to one side of the film membrane, as illustrated in FIGS. 4 and5. The adhesive spray would then be dried at controlled temperatures andtime to evaporate off a portion, preferably a majority, of adhesivesolvent carrier at which time the indicator (hexamethoxy red orheptamethoxy red) in an indicator solvent carrier is sprayed onto thesurface of the drying adhesive in such a time, temperature andconcentration to immobilize the indictor on the adhesive. The assemblythen moves on to a second controlled temperature and time process topromote the evaporation of the indicator solvent carrier. Once anappropriate drying time and temperature are achieved, the outer or innerlayer is then laminated to the immobilized indicator and adhesive with aslight pressure applied through the assembly rollers to assure completelamination without wrinkles. Finally a release can be applied to theproduct to maintain the integrity of the laminated film and sandwichassembly. The final film sandwich product can be made ready for furtherpackaging or wound dressing assembly.

In some embodiments the polymeric indicator film is for application as afood wrap. In some embodiments of the food wrap application, the widthof the source roll and the finished film roll can be approximatelytwelve (12) inches to up to about 84 inches. In some embodiments, thefinished polymeric indicator film can be collected and wound around thecollection roll with the top side or interior side to the outside of thecollection roll as seen in FIG. 4. In some embodiments, the finishedproduct roll of film can be collected on and wound around the collectionroll with the top side or interior side to the inside of the collectionroll as seen in FIG. 5.

In some embodiments, a release liner may be introduced as a secondlaminate process just prior to the line being wound onto the finishedfilm roll. In some embodiments, a release liner can be employed foradded separation of the film as it is wound onto the collection roll.

The foregoing and other aspects of the embodiments disclosed herein maybe better understood in connection with the following examples.

EXAMPLES

In the examples below as well as throughout the application, thefollowing abbreviations have the following meanings. If not defined, theterms have their generally accepted meanings.

-   -   ° C.=degrees Celsius    -   DE=Diatomaceous earth    -   ° F.=Degrees Fahrenheit    -   g=Gram    -   IPA=Isopropyl Alcohol    -   kg=Kilogram    -   L=Liter    -   M=Molar    -   ° C.=Degrees Celsius    -   mbar=Millibar    -   mg=Milligram    -   min=Minutes    -   mL=Milliliter    -   MW=Molecular Weight    -   m/z=Mass/Charge    -   PE=Polyethylene    -   PVOH=Polyvinyl Alcohol    -   RT=Room Temperature    -   w/w=Weight to weight

Example 1 Preparation of Heptamethoxy Red in Gram Scale Step 1:Synthesis of Methyl 2,4,6-trimethoxybenzoate (CAS 29723-28-2)

2,4,6-trimethoxybenzoic acid (CAS 570-02-5) (5.61 g, 26.42 mmol) wassuspended in 20 mL of methanol (CAS 67-56-1). Concentrated sulfuric acid(CAS 7664-93-9) (1 mL) was added to the mixture, and the reaction heatedto reflux for 24 hrs. The reaction was cooled to room temperature, andthe methanol (CAS 67-56-1) removed in vacuo. The residues were taken upin 50 mL 5% NaHCO₃ (CAS 144-55-8) and extracted with hexane (CAS110-54-3) until all the solids had dissolved. The hexane extract wasdried over anhydrous Na₂SO₄ (CAS 7757-82-6), filtered, and rotovapped todryness to give the desired product, methyl 2,4,6-trimethoxybenzoate(CAS 29723-28-2), as a white crystalline solid.

Step 2: Synthesis of Heptamethoxy Red

1-bromo-2,4-dimethoxybenzene (CAS 17715-69-4) (4.23 g, 19.47 mmol) wasadded to a round bottom flask, and the flask flushed with nitrogen for10 minutes. Anhydrous ether (CAS 60-29-7) (80 mL) was added, followed bythe drop wise addition of n-butyllithium (CAS 109-72-8) in hexane (CAS110-54-3) (1.6M, 12.2 mL). The cloudy mixture was stirred at roomtemperature for 10 minutes. Methyl 2,4,6-trimethoxybenzoate (CAS29723-28-2) (2.20 g, 9.74 mmol) was dissolved in ether (CAS 60-29-7),and added drop wise to the reaction mixture. After the addition wascomplete, the reaction was stirred for 3 minutes longer. The reactionwas then poured into a separatory funnel containing 5% NH₄Cl (CAS12125-02-9) (50 mL) and shaken until a color change was observed. Thelayers were separated, and the ether layer was dried over anhydrousNa₂SO₄ (CAS 7757-82-6), filtered, and rotovapped to dryness. The crudeoil was placed in the freezer. (6.02 g, 132% due to impurities).

Example 2

One Step Preparation of Heptamethoxy Red

Add (4.23 g, 19.47 mmol) 1-bromo-2,4-dimethoxybenzene (CAS 17715-69-4)to an appropriately sized round bottom flask. Attach a rubber septum toseal the flask.

Insert a needle into the septum as a vent and flush the round bottomflask with nitrogen for about 10 minutes.

Add (80 mL) anhydrous ether (CAS 60-29-7), followed by the drop wiseaddition of n-butyllithium (CAS 109-72-8) in hexane (CAS 110-54-3)(1.6M, 12.2 mL).

Stir the cloudy mixture for 10 minutes and keep the round bottom flaskon ice.

Dissolve (2.20 g, 9.74 mmol) of methyl 2,4,6-trimethoxybenzoate (CAS29723-28-2) in about 20 ml of anhydrous ether (CAS 60-29-7) (more than˜20 mL can be used if needed), and then add this drop wise to thereaction mixture.

After the addition is complete, stir the reaction mixture for about 3minutes longer.

Pour the reaction mixture into a separatory funnel containing 5% NH₄Cl(aq) (CAS 12125-02-9) (50 mL) and shake until a color change is observed(pale orange).

The layers are allowed to separated, and dry the top ether layer withabout 5 g anhydrous Na₂SO₄ (CAS 7757-82-6), filter, and rotovapped todryness at 35-40° C. under 400 mbar.

Place the crude oil of heptamethoxy red (yellow-orange in color) intothe freezer.

Yield is ˜3.1 g.

Example 3 Preparation of Hexamethoxy Red in Gram Scale

Add (4.23 g, 19.47 mmol) 1-bromo-2,4-dimethoxybenzene (CAS #17715-69-4)to an appropriately sized round bottom flask.

Attach a rubber septum to seal the flask.

Insert a needle into the septum as a vent and flush the round bottomflask with nitrogen for about 10 minutes.

Add (80 mL) anhydrous ether (CAS #60-29-7), followed by the drop wiseaddition of n-butyllithium (CAS #109-72-8) in hexane (CAS #110-54-3)(1.6M, 12.2 mL).

Stir the cloudy mixture for 10 minutes and keep the round bottom flaskon ice.

Dissolve (2.20 g, 9.74 mmol) of methyl 2,4-dimethoxybenzoate (CAS#2150-41-6) in about 20 ml of anhydrous ether (CAS #60-29-7) (more thanabout 20 ml can be used if needed), and then add this drop wise to thereaction mixture.

After the addition is complete, stir the reaction mixture for about 3minutes longer.

Pour the reaction mixture into a separatory funnel containing 5% NH₄Cl(aq) (CAS #12125-02-9) (50 mL) and shake until a color change isobserved (pale orange).

The layers are allowed to separated, and dry the top ether layer withabout 5 g anhydrous Na₂SO₄ (CAS #7757-82-6), filter, and rotovapped todryness at 35-40° C. under 400 mbar.

Place the crude oil of hexamethoxy red (yellow-orange in color) into thefreezer.

Yield is about 3.1 g.

Example 4 Heptamethoxy Red in DE for Addition to Blown ExtrudedPolyethylene Film

The following procedure describes the steps for incorporating 1%Heptamethoxy Red (HMR) via a 3% DE (DE=diatomaceous earth in PE pellets)load into about 200 feet of blown extruded PE (polyethylene) film.

The extruded PE film is about one (1) mil (0.001″) thick×48″ wide with aDE load of up to 3% and about 1% Heptamethoxy Red in this example.

Step 1: Dilute 20 g of HMR with 40 g of ethanol to get a 33% solution ofHMR in ethanol (20 g HMR+40 g ethanol=60 g of a 33% solution of HMR inethanol).

Step 2: Take 60 g of a 33% solution of Heptamethoxy Red in ethanol andadd it to 60 g of DE in a beaker on a hot plate over very lowtemperature using a stir bar and stir plate. Mix and heat slightly untilthe 40 g of ethanol evaporates leaving behind the 60 g of DE and 20 g ofHeptamethoxy Red (therefore, gives 80 g of a 25% concentrationHeptamethoxy Red in DE).

Step 3: Add the 80 g of the DE (containing a 25% concentration ofHeptamethoxy Red) to the blown extrusion PE film line via thegravimetric fillers at a rate of 4% (w/w) which will provide for 1% HMRand 3% DE in the final blown PE film. Switch the gravimetric filler “On”which introduces the 4% (w/w) DE to the PE and within three minutes theblown extruded PE film coming out at the end of the blown extruder willhave incorporated into it 1% HMR and 3% DE.

Example 5 Food Storage Bag Adaptation

This example illustrates a food storage bag for detecting the presenceof bacterial metabolic byproducts.

Bag product manufacture and materials: The polymeric indicator of thisinvention may be manufactured as a bag or as part of a bag.

Bag product configuration for closure: The bag may be of a zip-locktype, or have another closure system allowing moisture to be trappedwithin the bag, so as to preserve the moisture of the food stuff.

Bag product size: Although any bag size may be used, this example is toillustrate a particular configuration for consumer use. Forconsumer-bagged spinach, a 48 ounce bag, optionally resealable, isfabricated. The bag may have optional clasps to reduce the inner volumeif the consumer does not use the entire product at once. This will havethe effect of concentrating any microbial byproducts which may bepresent or have arisen after the original opening of the bag. Localconcentration of the microbial byproduct may have the ultimate effect ofproducing a stronger visual signal as the chromophores may be morereadily available for ionic saturation.

Example 6 Polyethelene/Polyurethane Laminate

This example illustrates a polyethelene/polyurethane laminate joined byan adhesive containing a pH indicating moiety.

To a polyethylene sheet (Glad® Wrap) was sprayed Elmer's Multi-PurposeSpray Adhesive, 4 ounce, No E452. The spray adhesive dried inapproximately five minutes after application and left a thin transparentfilm of the spray on the Glad® Wrap. The adhesive was clear after dryingand did not have a negative effect on the flexibility characteristics ofthe Glad® Wrap film. The heptamethoxy red indicator in ethanol (5 mL)was lightly sprayed over the adhesive layer using a spray bottle,immobilizing the indicator on the surface of the adhesive. The compositewas allowed to dry overnight.

The polyurethane films Tegaderm (3M) and 1 mil DT 1001 84 Shore APolyether Polyurethane film (American Polyfilm Inc., Banford, Conn.)were used in the tests. The polyurethane film was applied to thepolyethylene/adhesive/indicator assembly using a rolling pin. Dilute HClwas applied to the polyurethane films, resulting in a bright colorchange from colorless to violet red taking place slowly over a period of12 to 16 hours.

Example 7 Synthesis of Polyethylene Film with Hexamethoxy Red

The following example demonstrates how to prepare a polyethylene filmwith hexamethoxy red associated therewith.

In this example, from 1-3% (w/w) PE/DE in pellet form is added to ablown extrusion line making a PE film that is 0.001 inch thick×48 incheswide. The blown extrusion process is adjusted such that it takes aboutten (10) minutes for the gravimetrically fed PE/DE pellets entering theheated auger at the front end of the process to thoroughly mix with thePE and produce a 0.001 inch blown PE film coming out the back end of theprocess.

Line speed is adjusted and set to produce 120 feet of PE film per minute(at 0.001 inch thick×48 inches wide).

The amount of PE used during this production run will be about 1.0 kgper minute (or about 1.0 kg per 120 feet of extruded film) whenfollowing the above specifications for line speed, film thickness andfilm width. Dwell temperature for the ten (10) minutes in the auger is≦420° F. (215° C.) and the hexamethoxy red is heat stable for thisperiod of time at this temperature.

The PE blown extrusion process will start their normal supply of PE(with the gravimetric filler in the “Off” position). The gravimetricfeeder will then be supplied with 700 g of PE/DE comprising sufficientamounts of hexamethoxy red so that the final concentration is 1% (w/w)and is set to add 1% (w/w) of the PE/DE to the PE being blown extruded.

Once the blown extrusion process is normalized and producing acceptablePE film, a switch is made to the gravimetric filler to the “On” positionwhich begins to introduce 1% (w/w) PE/DE to the PE film and within aboutten (10) minutes the blown extruded PE film coming out at the end of thepilot line will have incorporated into it the addition of the 1% (w/w)PE/DE.

2% (w/w) and 3% (w/w) compositions are likewise prepared.

Example 8 Synthesis of Polyurethane or Polystyrene Film with HexamethoxyRed

The process of Example 7 is repeated except that PE is replaced withpolyurethane (PU), polystyrene (PS) or any one of or combination of anumber of different polymers where each polymer may provide a desiredfunctionality.

Example 9 Preparation of a Polymeric Indicator Film as a Food Wrap

As shown in the FIG. 4 the food warp manufacturing process begins with ahydrophobic, water impermeable film 3, for example a polyethylene filmsuch as Glad® wrap, which is the outer layer of the polymeric indicatorfilm. The characteristic of the hydrophobic, water impermeable film 3 isits resistance to the passage of environmental and ambient gaseouscompounds such as oxygen, hydrogen, nitrogen, moisture or otherelements. The film 3 is brought to an adhesive spraying process 12, forexample by the movement of the first spindle 20, where it is sprayedwith an adhesive, preferably in a light spray or mist form. The adhesivemay comprise one or more compounds in an aerosol. The film is then movedthrough the adhesive spraying process 12 to a first evaporation process13 where an initial drying process of the adhesive begins. The adhesivemay have a drying time of about five minutes at ambient temperatures andnormal humidity conditions. The drying process may be accelerated by theaddition of heat and/or forced airflow. The amount of drying can becontrolled by the speed of the film through the first evaporationprocess 13, the temperature of the heat applied and/or the amount offorced air moved across the surface. The objective is to initiateevaporation of the carrier of the adhesive, such as acetone, whileallowing the residual adhesive to accept the indictor, for example, adiluted heptamethoxy red used for the food warp application. The heatand/or forced air applied should be limited so as not to adverselyaffect the outer layer and/or the adhesive. A fume hood can be providedto collect the carrier vapors in a safe manner and dispose of the samein accordance with environmental regulatory requirements.

After the film has passed through the first evaporation process 13, itgoes to the indicator spraying process 14 in which the adhesive receivesan application of the indicator, for example, a dilute heptamethoxy redin a carrier, such as heptamethoxy red in 95% ethanol. The indicator ispreferably applied in a light spray or mist form. The indicatorheptamethoxy red in ethanol is applied to the surface of the dryingadhesive in such a manner as to have the indicator immobilized on thesurface of the drying adhesive. The film then passes though a secondevaporation process 15 for evaporation of the carrier of the indicator.Heat and/or forced air may be applied to assist in the evaporation. Theheat and/or forced air applied should be limited so as not to adverselyaffect the outer layer, the adhesive and/or the indicator. Once again afume hood may be provided to collect the carrier vapors in a safe mannerand dispose of the same in accordance with environmental regulatoryrequirements. Both the adhesive and the indicator are in the dryingcondition as the process moves into the lamination process 16. In thelamination process 16, a hydrophilic, hydronium ion penetrating film 9,which is the inner layer of the polymeric indicator film and may besupplied by the second spindle 21, is applied in a conventional methodwhich carefully and gently presses the two layers together, for exampleby operation of rollers 22. The hydrophilic, hydronium ion penetratingfilm 9 may be a film with a relative high Moisture Vapor TransmissionRate (MVTR) such as Pebax® MV 3000 film. The residual adhesive should besufficient to provide added laminate qualities to the film and containthe assembly as a “sandwich” containing the adhesive and immobilizedindicator between the inner and outer layers. A second laminate processusing a release liner, which is not shown in FIG. 4, can be applied tothe film(s) just prior to collection on a finished polymeric indicatorfilm 1 by the third spindle 23.

Example 10 Preparation of a Polymeric Indicator Film for MedicalApplication

As shown in FIG. 5 the manufacturing process of a polymeric indicatorfilm for medical application begins with hydrophobic, water impermeablefilm 3. Preferably the hydrophobic, water impermeable film 3 used in amedical application is characterized by its passage of environmental andambient gaseous compounds through the “barrier” such as oxygen,hydrogen, and nitrogen. The porosity of film 3 is such that it isresistant to the passage of a dipole molecule such as water. Forexample, film 3 may be a polyurethane film similar to that used by 3M™in its Tegaderm™ product. Film 3 is brought to an adhesive sprayingprocess 12, for example by the movement of the first spindle 20, whereit is first sprayed with an adhesive, preferably in a light mist orspray form. The adhesive may comprise one or more compounds in anaerosol. The film then is moved through the adhesive spraying process 12and on to a first evaporation process 13 where an initial drying processof the adhesive begins. The adhesive may have a drying time of aboutfive minutes at ambient temperatures and normal humidity conditions. Thedrying process may be accelerated by the addition of heat and/or forcedairflow. The amount of drying can be controlled by the speed of the filmthrough the first evaporation process 13, the temperature of the heatapplied and/or the amount of forced air moved across the surface. Theobjective is to initiate evaporation of the carrier while allowing theresidual adhesive to accept the indictor, for example, a dilutedhexamethoxy red used for the medical application. The heat appliedshould be limited so as not to adversely affect the outer layer and/orthe adhesive. A fume hood can be provided to collect the carrier vaporsin a safe manner and dispose of the same in accordance withenvironmental regulatory requirements.

After the film has passed through the first evaporation process 13, itgoes to the indicator spraying process 14 in which the adhesive receivesan application of the indicator, for example, a dilute hexamethoxy redin a carrier, such as ethanol. The indicator is preferably applied in alight spray or mist form. The indicator hexamethoxy red spray in ethanolis applied to the surface of the drying adhesive in such a manner as tohave the indicator immobilized on the surface of the drying adhesive.The film then passes though a second evaporation process 15 to assist inthe evaporation of the carrier of the indicator. Heat and/or forced airmay be applied to assist in the evaporation. The heat and/or forced airapplied should be limited so as not to adversely affect the outer layer,the adhesive and/or the indicator. Once again a fume hood may beprovided to collect the carrier vapors in a safe manner and dispose ofsame in accordance with environmental regulatory requirements. Both theadhesive and the indicator are in the drying condition as the processmoves into the lamination process 16. During the lamination process 16,hydrophilic, hydronium ion penetrating film 9, which is the inner layerof the polymeric indicator film and may be supplied by the secondspindle 21, is applied in a conventional method which carefully andgently presses the two films together, for example by operation of therollers 22. The film 9 may be a film with a relative high Moisture VaporTransmission Rate (MVTR) such as Pebax® MV 3000 film or a polyurethanefilm with a high MVTR. The residual adhesive should be sufficient toprovide added laminate qualities to the film and contain the assembly asa “sandwich” containing the adhesive and immobilized indicator betweenthe inner and outer layers. A second laminate process using a releaseliner, which is not shown in FIG. 5, can be applied to the film(s) justprior to collection on finished polymeric indicator film 1 by the thirdspindle 23.

Additional rollers, which are not shown in FIGS. 4 and 5, may be used tosupport the film from above and/or below as it moves through thesequenced series of applications. These rollers can be employed not onlyto support the film from above and/or below but to mitigate anywrinkling of the film during the manufacturing processes.

Example 11 Application of a Polymeric Indicator Film on Food

A polymeric indicator film was each applied to a sample of ground beefcontaminated with Escherichia coli (E. coli) of less than 100 colonyforming units (CFUs) and to a sample of chicken contaminated withSalmonella of less than 100 CFUs at ambient conditions. Red colorappeared on a significant portion of the polymeric indicator film within18 hours. The following table summarized the results.

Color of the film Color of the film 18 at the time it was hours after itwas Food applied on the food applied on the food beef contaminated clearDeep red on a significant with Escherichia coli portion of the filmchicken contaminated clear Deep red on a significant with Salmonellaportion of the film

The embodiments and examples described above are not intended to limitthe invention. It should be understood that numerous modifications andvariations are possible in accordance with the principles of the presentinvention.

1. A polymeric indicator film comprising a transparent polymer layer orlayers and a plurality of pH indicating moieties, wherein the pHindicating moieties are entrapped within the polymer layer or betweentwo or more of the polymer layers and further wherein the pH indicatingmoieties retain the transparency of the polymer film at neutral pH butwhich impart color to at least a portion of the film when exposed to anacidic pH.
 2. A polymeric indicator film comprising a polymer and aplurality of pH indicating moieties selected from heptamethoxy red andhexamethoxy red or a combination thereof, wherein the pH indicatingmoieties are entrapped within the polymer.
 3. A polymeric indicator filmcomprising: a) a first layer comprising one or more hydrophilic,hydronium ion penetrating layers; b) a second layer comprising one ormore hydrophobic, water impermeable layers; and c) a pH indicator layerplaced between the first layer and the second layer.
 4. The polymericindicator film of claim 1 wherein the polymer is selected from the groupconsisting of polyethylene terephthalate, poly(vinylidene fluoride),poly(vinyl chloride), poly(vinylidene chloride), polypropylene, phenoxyresins, butadiene/styrene copolymers, butadiene/methylstyrenecopolymers, poly(meth)acrylates, butadiene/acrylonitrile copolymers,ethylene/propylene copolymers, polybutadiene, polyisoprene,poly(oxy-2,6-dimethyl-1,4-phenylene),poly(oxycarbonyloxy-1,4-phenyleneisopropylidene-1,4-phenylene),acrylonitrile styrene copolymers, acrylonitrile/methylacrylate/butadiene copolymers, acrylonitrile/styrene/butadienecopolymers, poly-1-vinylnaphthalene, polyvinylphenyl ketone,poly-p-xylenedodecanedioate, poly-tetramethylene octenediamide,poly-tetramethylene terephthalene, poly-trimethylene-3,3′-dibenzoate,poly-terephthallic anhydride, poly-4-methyl-diamine, polyvinylenecarbonate, polyvinylene laurate, polyisopropenyl acetate,polyallylbenzene, polyvinylbutyl ether, polyvinyl formate, polyvinylphenyl ether, polynorbornadine, polycarbonate, hydrophobic polyestersand polyurethanes, or a mixture thereof.
 5. The polymeric indicator filmof claim 1 wherein the polymer is polyethylene.
 6. The polymericindicator film of claim 1 that is transparent.
 7. The polymericindicator film of claim 1 that undergoes a detectable colorimetricchange in the presence of an acid.
 8. The polymeric indicator film ofclaim 7 wherein the acid is selected from the group consisting ofcarbonic acid, sulfuric acid, and hydrogen sulfide.
 9. The polymericindicator film of claim 7 wherein the acid is a microbial byproduct oris formed by reaction of a microbial byproduct with water, saidmicrobial byproduct selected from the group consisting of carbon dioxideand sulfur dioxide.
 10. The polymeric indicator film of claim 9 whereinthe microbial byproduct is generated by a microbe selected from thegroup consisting of Bacillus, Brucella, Campylobacter, Clostridium,Escherichia coli, Listeria monocytogenes, Salmonella, Streptococcus,Pseudomonas aeruginosa, Staphylococcus aureus, Shigella spp., Vibrispp., Yersini spp. or a mixture of two or more such microbes.
 11. A foodstorage container containing the polymeric indicator film of claim 1.12. The food storage container of claim 11 that is a sealable bag. 13.The food storage container of claim 11 that is a jug or bottle forstoring liquids.
 14. A flexible pH indicating wrap comprising an outerhydrophobic barrier layer and an indicator layer, wherein the indicatorlayer is the polymeric indicator film of claim
 1. 15. A flexible pHindicating wrap comprising an outer hydrophobic barrier layer and aninner barrier layer permeable to microbial byproducts and acids thereof,wherein sandwiched between the outer and inner layers is a plurality ofpH indicating moieties selected from heptamethoxy red and hexamethoxyred or a combination thereof, and wherein the pH indicating moieties areentrapped within a polymer or are adherent to an adhesive, said polymeror adhesive being situated between the outer and inner layers.
 16. Theflexible pH indicating wrap of claim 15 wherein the outer and innerlayers are independently selected from the group consisting ofpolyethylene, polyethylene terephthalate, poly(vinylidene fluoride),poly(vinylidene chloride), phenoxy resins, butadiene/styrene copolymers,butadiene/methylstyrene copolymers, poly(meth)acrylates,butadiene/acrylonitrile copolymers, ethylene/propylene copolymers,polybutadiene, polyisoprene, poly(oxy-2,6-dimethyl-1,4-phenylene),poly(oxycarbonyloxy-1,4-phenyleneisopropylidene-1,4-phenylene),acrylonitrile styrene copolymers, acrylonitrile/methylacrylate/butadiene copolymers, acrylonitrile/styrene/butadienecopolymers, poly-1-vinylnaphthalene, polyvinylphenyl ketone,poly-p-xylenedodecanedioate, poly-tetramethylene octenediamide,poly-tetramethylene terephthalene, poly-trimethylene-3,3′-dibenzoate,poly-terephthallic anhydride, poly-4-methyl-diamine, polyvinylenecarbonate, polyvinylene laurate, polyisopropenyl acetate,polyallylbenzene, polyvinylbutyl ether, polyvinyl formate, polyvinylphenyl ether, polynorbornadine, polycarbonate, hydrophobic polyestersand polyurethanes, or a mixture thereof.
 17. The flexible pH indicatingwrap of claim 14 having attached thereto or printed thereon a machinerecognizable code.
 18. The flexible pH indicating wrap of claim 17wherein the code is a barcode.
 19. The flexible pH indicating wrap ofclaim 18 wherein the code is in a RFID (radio frequency identification)tag.
 20. A method for detecting whether food is spoiled or contaminatedwith microbes such that said food is not edible, said method comprising:a) placing a portion of said food proximal to the polymeric indicatorfilm of claim 1; b) detecting the presence or absence of a colorimetricchange in the polymeric indicator film or pH indicating wrap; and c)correlating the presence or absence of a colorimetric change in thepolymeric indicator film or pH indicating wrap to whether the food isnon-edible or edible. 21-38. (canceled)
 39. A process for preparing apolymeric indicator film comprising an inner hydrophilic, hydronium ionpenetrating transparent layer and an outer hydrophobic, waterimpermeable transparent layer which process comprises: a) selecting oneor more hydrophilic, hydronium ion penetrating layers as the inner layerof the polymeric film wherein the inner layer has a first and secondsurface; b) selecting one or more hydrophobic, water impermeable layersas the outer layer of the polymeric film wherein the outer layer has afirst and second surface; c) applying a pH indicator layer to at least aportion of one surface of the outer layer; and d) bonding the inner andouter surfaces together such that the pH indicator layer is placedbetween the inner and outer layers.
 40. A process for preparing apolymeric indicator film comprising an inner hydrophilic, hydronium ionpenetrating transparent layer and an outer hydrophobic, waterimpermeable transparent layer which process comprises: a) selecting oneor more hydrophilic, hydronium ion penetrating layers as the inner layerof the polymeric film wherein the inner layer has a first and secondsurface; b) selecting one or more hydrophobic, water impermeable layersas the outer layer of the polymeric film wherein the outer layer has afirst and second surface; c) applying an adhesive layer to one surfaceof the outer layer; d) applying a coating of pH indicator to at least aportion of the adhesive layer; and e) bonding the inner and outersurfaces together such that the adhesive layer coated with the pHindicator is placed between the inner and outer layers.
 41. The processof claim 40, wherein the adhesive is partially dried before the coatingof pH indicator is applied.
 42. The process of claim 40, wherein theouter layer with the indicator applied thereto is dried before bindingof the outer layer with the inner layer.
 43. The process of claim 39,wherein the pH indicator is selected from the group consisting ofxylenol blue (p-xylenolsulfonephthalein), bromocresol purple(5′,5″-dibromo-o-cresolsulfonephthalein), bromocresol green(tetrabromo-m-cresolsulfonephthalein), cresol red(o-cresolsulfonephthalein), phenolphthalein, bromothymol blue(3′,3″-dibromothymolsulfonephthalein), p-naphtholbenzein(4-[alpha-(4-hydroxy-1-naphthyl)benzylidene]-1(4H)-naphthalenone),neutral red (3-amino-7-dimethylamino-2-methylphenazine chloride),hexamethoxy red and heptamethoxy red, and combinations thereof.
 44. Theprocess of claim 39, wherein the pH indicator comprises hexamethoxy red.45. The process of claim 39, wherein the pH indicator comprisesheptamethoxy red.
 46. The process of claim 39, wherein the polymericindicator film is used as a food wrap.
 47. The process of claim 39,wherein the polymeric indicator film is used in a medical setting.